NZB mice are genetically predisposed to developing anti-erythrocyte autoantibodies resulting in the development of Coombs-positive hemolytic anemia. Despite the use of this model of the study of organ-specific autoimmunity for 3 decades, little structural information has been obtained on pathogenic anti-erythroycyte autoantibodies or on the epitopes recognized by these antibodies. Indeed, few pathogenic autoantibodies with specificity for any self antigen have been characterized at the molecular level. Although autoantibodies have been detected that react serologically with a wide variety of "self" antigens including DNA and bromelain-treated erythrocytes, it is now apparent that most such antibodies are not pathogenic. Our overall hypothesis is that pathogenic autoantibodies may represent a restricted subset of the total repertoire of serologically defined autoantibodies. In preliminary studies, we have shown that autoantibodies eluted from Coombs-positive erythrocytes may exhibit a highly restricted isoelectric focusing pattern. Furthermore, we have shown evidence that the spontaneous autoantibody response of NZB mice to self- erythrocytes consists of antibodies that are similar in specificity and idiotype expression to a pathogenic monoclonal antibody (G8) that was "cloned" from an autoimmune NZB mouse. Our first specific aim is to extend these observations utilizing a more complete panel of hybridomas to test the hypothesis that pathogenic anti-erythrocyte antibodies may be restricted in immunoglobulin V gene usage, isoelectric focusing patterns and idiotype expression. In other preliminary studies, we have sequenced the VH and VL regions of the G8 (anti-MRBC) autoantibody and found that the sequence of the VH region is nearly identical to that of an anti-DNA mAb from autoimmune-prone MRL mice. In addition, both antibodies are similar to a germline gene (VH6) derived from a normal mouse strain. These results suggest that a single germline VH gene may encode autoantibodies with two distinct specificities. Very little is known about germline V genes from autoimmune-prone NZB mice, therefore, it is imperative to analyze the relevant genes at the molecular level. The second specific aim will be to obtain the nucleotide sequences of the V regions encoding this panel of pathogenic anti-erythrocyte autoantibodies and to determine their relationship to germline V genes. Conservation of autoantibody V genes in the germline would provide evidence against the somatic mutation hypothesis of the generation of autoimmunity and would strengthen the view that a pathogenic autoimmune response results from disregulation of specific clones of potentially autoimmune lymphocytes. Information gained in this study will provide a better understanding of autoimmune responses at the clonal and molecular levels and may open up new approaches towards specific therapy of autoimmune diseases including systemic lupus erythematosus, glomerulonephritis, and rheumatoid arthritis.